The invention relates to novel bacterially-produced antimicrobial peptides; more particularly the invention relates to a dehydroalanine-containing lantibiotic.
Lantibiotics are bacterially-produced antimicrobial peptides that possess unique chemical and biological properties owing to their containing a variety of unusual amino acid residues. Lantibiotics are defined as such by the presence of lanthionine or xcex2-methyllanthionine, which are introduced by a posttranslational process in which serine or threonine is dehydrated to the corresponding dehydro residue, which then reacts in a Michael-type addition of a cysteine sulfhydryl group to the double bond of the dehydro residue to form a thioether link [reviewed in (1-6)]. Mature lantibiotics typically contain one or more dehydro residues that do not participate in lanthionine bridges. The unique properties that are conferred by these unusual residues results in their being useful components in the design of novel biomolecules (1,2,7,8).
One of the attractive features of lantibiotics is that they are comprised of gene-encoded polypeptide sequences, so their structures can be manipulated by protein engineering. Whereas this is simple in concept, putting it into practice requires the utilization of many different genetic and recombinant DNA techniques, including the removal and replacement of chromosomal segments with their genetically-engineered counterparts. Ideally, these manipulations need to be done in such a way that the engineered lantibiotic analog be efficiently produced so that useful amounts of the analog are available for experimentation, which implies a need to engineer regulatory elements. Only a few bacterial strains have been sufficiently characterized to permit these manipulations to be performed in a convenient and facile manner. One such well-characterized bacterial strain is Bacillus subtilis 168, which is second only to E. coli in the extent to which tools of genetic and protein engineering have been developed, which has contributed to the extensive use of B. subtilis 168 for the industrial production of bio-engineered materials. The advantage of B. subtilis 168 over other bacterial strains has recently been increased even more by the availability of the complete sequence of the B. subtilis 168 genome (9).
The present inventor has discovered a new lantibiotic, named sublancin 168, that is a natural product produced by B. subtilis 168. Although approximately twenty lantibiotics are already known, the fact that this new lantibiotic is indogenous to B. subtilis 168, and thus can be studied and manipulated using the powerful methods that are available in this strain, suggests that progress in our understanding of lantibiotics will be accelerated by our ability to study and manipulate sublancin and the genes associated with its production in its natural B. subtilis 168 host. In addition to this practical aspect of the discovery, sublancin 168 has structural features and physical properties, such as the presence of disulfide bridges and extraordinary stability, that are unprecedented among the known lantibiotics.
Therefore, the present invention is directed to a peptide having an amino acid sequence which is at least 80% identical with SEQ ID NO: 7 prior to dehydration of serines and threonines and formation of thioether cross-linkages.
The invention also is directed to a peptide having an amino acid sequence which is at least 80% identical with SEQ ID NO: 5 prior to dehydration of serines and threonines and formation of thioether cross-linkages.
The invention is further directed to a peptide having an amino acid sequence which is at least 80% identical with SEQ ID NO: 18.
The peptides of the invention may be incorporated into a pharmaceutical preparation suitable for treating a bacterial infection. In addition, a bacterial-growth-inhibiting effective amount of one or more of the peptides of the invention may be added to a food for preservation against bacteria-mediated spoilage of the food.
The invention also includes a DNA which has a nucleic acid sequence which is at least 80% identical with SEQ ID NO: 4, preferably at least 80% identical with nucleotides 219-389 or nucleotides 447-782 of SEQ ID NO: 4, which includes the genetic sequence encoding the sublancin 168 peptide of the invention. More preferably, the DNA has a nucleic acid sequence which is at least 80% identical with nucleotides 219-389 of SEQ ID NO: 4.
The invention further includes a composition suitable for killing or inhibiting growth of bacteria, comprising the peptides of the invention and a carrier. Another composition encompassed by the invention comprises the peptides of the invention and a second active agent selected from the group consisting of a lantibiotic (such as nisin or subtilin) and an antibiotic.